the twin-arginine translocation (tat) system is essential ... · the twin-arginine translocation...
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The twin-arginine translocation (Tat)system is essential for Rhizobium-legume
symbiosisS. Meloni, et al. 2003. Molecular Microbiology 48(5): 1195-1207
Presented by Denise AslettMicrobiology Journal Club
March 19, 2007
Rhizobium leguminosarum
• _-proteobacteria
• Symbiont of legumes
• 3 biovars that differ in hostspecificity– viciae (peas and broad
beans)
– trifolii (clovers)
– phaseoli (kidney beans)
• Host specificity genescarried on large plasmidsthat also carry genes fornitrogen fixation
• tat genes on chromosomeSource: Genome Biology 2006, 7:R34
Twin-arginine Transporter (Tat)
Lee, P., Tullman-Ercek, D. and Georgiou, G. 2006. The Bacterial Twin-Arginine Translocation Pathway. Annual Review of Microbiology. 60:373-395.
Ser/Thr-Arg-Arg-x-Phe-Leu-Lys, where x = polar aa
Rhizobium-Legume Symbiosis Overview
Sources: 1) Legume-Rhizobium Symbiosis from http://quorumsensing.ifas.ufl.edu/HCS200/LegRhiz.html, and 2) Gage, D. J. and Margolin, W. 2000. Hanging by athread: invasion of legume plants by rhizobia. Current Opinion in Microbiology. 3:613-617.
Hypothesis and Experiments
• The Tat system is essential for Rhizobium-legume symbiosis.– Characterized tat gene cluster and analyzedtat expression
– Examined hydrogenase translocation andactivity
– Examined N2 fixation phenotype in a tatBCmutant
– Analyzed outer membrane integrity in a tatmutant
Table 1Hydrogenase activity in tat mutants is
significantly diminished
SPF25 = mutant expressing hydrogenase in vegetative cells
SP61 = Tat deficient mutant of SPF25
Figure 2Membrane targeting of Hydrogenasesubunits is disrupted in tat mutants
SPF25 SP61
mutantexpressing
hydrogenasein vegetative
cells =
Tatdeficientmutant ofSPF25 =
Hydrogenase structure fromDesulfovibrio gigas courtesy of
www.chem.ox.ac.uk/icl/faagroup/dgigasx.gif
Figure 3APlants inoculated with a tatBC mutant were
deficient in N2 fixation
Used to assessnitrogenase activity in
environmentalsamples
Acetylene Reduction Assay
UPM791=Wild type
SM61 =tatBC mutant
Small, round,white noduleslacking acetylenereduction activity
Pink, normalnodules expressinghigh levels ofacetylene reductionactivity (data notshown)
Left: chlorotic peas fromwww.soilhealth.com
Figure 3BPlants inoculated with a tatBC mutant had lowernumbers of infected cells and no infection threads
UPM791 SM61 Wild type tatBC mutants
Figure 3BPlants inoculated with a tatBC mutant had lower
numbers of infected cells
UPM791 SM61 Wild type tatBC mutants
Figure 4 - Rieske protein
• Forms theiron sulphurcomponent ofcytochromebc1 complex
• Has a Tatdependentsignal peptide
N- terminal sequences from different Rhizobiaceae
Table 2Effects of tat mutation on cytochrome bc1
functionality
“Nadi” test
Nadi reagents:
• _-naphthol + dimethyl-pphenylenediamine+ O2 indophenol blue + H2O
• Oxidatively converted to the blue dye by cytochrome c
Figure 3BSS113 mutant with cytochrome bc1
deficiency can still infect plant cells
Wild type tatBC mutant bc1 mutant UPM791 SM61 SS113
Figures 5A & 5BEffect of tat mutation on R. leguminosarum
outer membrane
Wild type tatBC mutant
Rel
ativ
e O
D60
0
Increasing SDS _
wt tatBCB
LPS fractions
Conclusions• Tat genes are constitutively expressed in R.
leguminosarum vegetative cells.
• The mutation of tatBC genes abolished membranetargeting of the large and small hydrogenase subunitsand the effect is at the post-translational level.
• Tat mutations interrupt the symbiotic process beforebacteroid release in the cortex and the observedphenotype cannot be fully explained by a cytochromebc1 deficiency.
• A functional Tat pathway is essential for biologicalnitrogen fixation by the Rhizobium-legume symbiosis.